Protein c derivatives

ABSTRACT

Novel human protein C derivatives are described. These derivatives have increased anti-coagulation activity, resistance to serpin inactivation, and increased sensitivity to thrombin activation compared to wild-type protein C and retain the biological activity of the wild-type human protein C. These derivatives will require either less frequent administration and/or smaller dosage than wild-type human protein C in the treatment of acute coronary syndromes, vascular occlusive disorders, hypercoagulable states, thrombotic disorders and disease states predisposing to thrombosis.

[0001] This invention relates to novel polynucleotidea, polypeptidesencoded by them and to the use of such polynuoleotides and polypeptides.More specifically, the inverntion relates to human protein C derivativeswith inicreased anti-coagulant activity, resistance to Berpininactivation, increased sensitivity to thrombin activation, or acombination thereof, when cosnpared to wild-type activated protein C; totheir production, and to phacoutical compositions comprising these humanprotein C derivatives.

[0002] Protein C is a serine protease and naturally occurringanti-coagulant that plays a role in the regulation of hemostasis byinactivating Factors V_(a) and VIII_(a) in the coabulation cascade.Human protein C is made in vivo as a single polypeptide of 461 aminoacid a. This polypeptide undergoes multiple post-translationalmodifications including, 1) cleavage of a 42 amino acid signal sequence;2) cleavage of lysine and arginine residues (positions 156 and 157) tomake a 2-chain inactive precursor or zymogen (a 155 amino acid residuelight chain attached via a disulfide bridge to a 262 amino acid residueheavy chain); 3) vitamin K-dependent carboxylation of nine glutamic acidresidues located within the amino-terminal 45 residues (gla-domain):and, 4) carbohydrate attachment at four sites (one in the light chainand three in the heavy chain). Finally, the 2-chain zymogen may beactivated by removal of a dodecapeptide at the N-terminus of the heavychain, producing activated protein C (aPC) possessing greater enzymaticactivity than the 2-chain zymogen.

[0003] Blood coagulation is a highly complex process regulated by thebalance between pro-coagulant and anti-coagulant mechanisms. Thisbalance determines a condition of either normal hemostasis or abnormalpathological thrombus generation and the progression, for example, ofcoronary thrombosis leading to acute coronary syndromes (ACS; e.g.unstable angina, myocardial infarction). Two major factors control thisbalance; the generation of fibrin and the activation and subsequentaggregation of platelets. Both processes are controlled by thegeneration of the enzyme thrombin, which occurs following activation ofthe clotting cascade. Thrombin, in complex with thrombomodulin, alsofunctions as a potent anti-coagulant since it activates protein Czymogen to aPC, which in turn inhibits the generation of thrombin. Thus,through the feedback regulation of thrombin generation via theinactivation of Factors Va and VIIIa, aPC functions as perhaps the mostimportant down-regulator of blood coagulation resulting in protectionagainst thrombosis. In addition, aPC has anti-inflammatory properties,and exerts profibrinolytic effects that facilitate clot lysis.

[0004] Various methods of obtaining protein C from plasma and producingprotein C, aPC and protein C/aPC polypeptides through recombinant DNAtechnology are known in the art and have been described. See e.g., U.S.Pat. Nos. 4,775,624 and 5,358,932. Despite improvements in methods toproduce aPC through recombinant DNA technology, aPC and derivativesthereof are difficult and costly to produce.

[0005] Unlike the zymogen protein C, activated protein C has anextremely short half-life. A major reason for the short half-life isthat blood levels of aPC are regulated by molecules known as serpins(Serine Protease Inhibitors), which covalently bind to aPC forming aninactive serpin/aPC complex. The serpin/aPC complexes are formed whenaPC binds and proteolytically cleaves a reactive site loop within theserpin; upon cleavage, the serpin undergoes a conformational changeirreversibly inactivating aPC. The serpin/aPC complex is then eliminatedfrom the bloodstream via hepatic receptors for the serpin/aPC complex.As a result, aPC has a relatively short half-life compared to thezymogen; approximately 20 minutes for aPC versus approximately 10 hoursfor human protein C zymogen (Okajima, et al., Thromb Haemost63(1):48-53, 1990).

[0006] Therefore, an aPC derivative exhibiting resistance to serpininactivation, while maintaining the desirable biological activities ofaPC (e.g., anticoagulant, fibrinolytic, and anti-inflammatoryactivities), provides a compound that has an increased plasma half-lifeand is effectively more potent than the parent compound, requiringsubstantially reduced dosage levels for therapeutic applications. Thepotency advantages are especially important in disease states in whichserpin levels are elevated.

[0007] Additionally, an aPC derivative exhibiting increasedanti-coagulant activity, while maintaining the other biologicalactivities of aPC (e.g., fibrinolytic, and anti-inflammatoryactivities), provides a compound that is effectively more potent thanthe parent compound, requiring substantially reduced dosage levels fortherapeutic applications.

[0008] Enhancement of human protein C calcium and membrane bindingactivity by site-directed mutagenesis of the gla-domain has beenreported by several investigator a, for example, Shen et al. (J Biol.Chem., 273(47) 31086-91, 1998.) and Shen et al. (Biochemistry, 36(51)16025-31, 1997). Through continued scientific experiments, analysis, andinnovation, the present inventors identified specific sites and modifiedtargeted amino acid residues in the gla-domain of the aPC molecule.Surprisingly, we ford increased anti-coagulant activity of the aPCderivative when specific amino acid substitutions were performed.Therefore, an aPC derivative exibiting increased anti-coagulantactivity, while maintaining the other biological activities of aPC(e.g., fibrinolytic, and anti-inflammatory activities), provides acompound that is effectively more potent than the palaent compound,requiring substantially reduced dosage levels for therapeuticapplications.

[0009] Furthermore, human protein C derivatives with increasedsensitivity to thrombin activation (hyper-activatable zymogens) areuseful as site-activated anti-thrombotic agents, as described, forexample, in U.S. Pat. No. 5,453,373 and in Richardson et al. (ProteinScience, 3:711-712, 1994). Such hyper-activatable zyraogens can also beconstructed to contain the gla-domain mutants and the serpin resistantderivatives described above. These derivatives have increasedanti-coagulant activity, resistance to serpin inactivation, andincreased sensitivity to thrombin activation when compared to wild-typehuman protein C.

[0010] Accordingly, the present invention describes novel human proteinC derivatives. These human protein C derivatives retain the importantbiological activity when compared to wild-type protein C and haveincreased anti-coagulant activity, resistance to serpin inactivation,and increased sensitivity to thrombin activation when compared towild-type human protein C. Other protein C derivatives of the presentinvention have increased sensitivity to thrombin activation andincreased anti-coagulant activity or increased sensitivity to thrombinactivation and resistance to serpin inactivation.

[0011] Therefore, these compounds provide various advantages, forexample, site-activation, less frequent administration and/or smallerdosages and thus a reduction in the overall cost of production of thetherapy. Thus, these compounds exhibit an advantage over current therapyin disease states of acute coronary syndromes such as unstable angina ormyocardial infarction.

[0012] The present invention provides a human protein C derivativecomprising SEQ ID NO: 1 wherein Asp at position 167 is substituted withPhe; Asp at position 172 is substituted with Lys and further comprisingat least one amino acid substitution selected from the group consistingof:

[0013] His at position 10, Ser at position 11, or Ser at position 12 areindependently substituted with any amino acid; Gln at position 32 issubstituted with Glu; Asn at position 33 is substituted with Asp or Phe;and, amino acids at positions 194, 195, 228, 249, 254, 302, or 316 aresubstituted with an amino acid selected from Ser, Ala, Thr, His, Leu,Lys, Arg, Asn, Asp, Glu, Gly, and Gln.

[0014] The present invention also provides recombinant DNA moleculesencoding the human protein C derivatives of the present invention, inparticular those comprising SEQ ID Nos: 9, 10, 11, and 12.

[0015] Another aspect of the present invention provides proteinsequences of these same human protein C derivatives, particularly thosecomprising SEQ ID NOS: 3, 4, 5, and 6, and the activated forms thereof.

[0016] The present invention comprises methods of treating acutecoronary syndromes such as myocardial infarction and unstable angina.

[0017] The present invention further comprises methods of treatingthrombotic disorders. Such disorders include, but are not limited to,stroke, abrupt closure following angioplasty or stent placement, andthrombosis as a result of peripheral vascular surgery.

[0018] The present invention comprises methods of treating vascularocclusive disorders and hypercoagulable states including: sepsis,disseminated intravascular coagulation, purpura fulminans, major trauma,major surgery, burns, adult respiratory distress syndrome,transplantations, deep vein thrombosis, heparin-inducedthrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagicfever, thrombotic thrombocytopenic purpura, and hemolytic uremicsyndrome.

[0019] Another aspect of the invention comprises treating the diseasesand conditions caused by or resulting from protein C deficiency asdefined herein.

[0020] Another embodiment of the present invention is a method oftreating sepsis comprising the administration to a patient in needthereof, a pharmaceutically effective amount of a human protein Cderivative of this invention in combination with bacterial permeabilityincreasing protein.

[0021] Another embodiment of the present invention is a method oftreating thrombotic disorders which comprises: administering to apatient in need thereof a pharmaceutically effective amount of a humanprotein C derivative of this invention in combination with ananti-platelent agent.

[0022] The present invention further provides a method of treating acutearterial thrombotic occlusion, thromboembolism, or stenosis in coronary,cerebral or peripheral arteries or in vascular grafts which comprisesadministering to a patient in need thereof a pharmaceutically effectiveamount of a human activated protein C in combination with a thrombolyticagent.

[0023] The present invention further provides a method of treating humanpatients with genetically predisposed prothrombotic disorders, forexample, protein C deficiency, Factor V Leiden mutation, and prothrombingene G20210A mutation, which comprises administering gene therapy tosaid patients with a recombinant DNA molecule encoding a protein Cderivative.

[0024] The present invention also provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and a humanprotein C derivative of this invention.

[0025] Methods and aspects of producing the novel isolated human proteinC derivatives are also an aspect of this invention.

[0026] The present invention also provides for the use of the humanactivated protein C derivatives of this invention for the manufacture ofa medicament for the treatment of the above-mentioned indications

[0027] Methods and aspects of producing the novel human proteinderivatives are also an aspect of this invention.

[0028] For purposes of the present invention, as disclosed and claimedherein, the following terms are as defined below.

[0029] Anti-platelet agent—one or more agents alone or in combinationwhich reduces the ability of platelets to aggregate. Agents understoodand appreciated in the art include those cited in, for example,Remington, The Science and Practice of Pharmacy, Nineteenth Edition, VolII, pages 924-25, Mack Publishing Co., herein incorporated by reference.Such agents include but are not limited to aspirin (ASA), clopidogrel,ReoPro® (abciximab), dipyridamole, ticlopidine and IIb/IIIa antagonists.

[0030] Zymogen—protein C zymogen, as used herein, refers to secreted,inactive forms, whether one chain or two chains of protein C orderivatives thereof. Cleavage of lysine and arginine residues (positions156 and 157) results in a 2-chain (heavy and light) inactive zymogen.

[0031] Activated protein C refers to the activated form of protein Czymogen which is produced after by removal of a dodecapeptide at theN-terminus of the heavy chain, producing activated protein C.

[0032] Activated protein C or aPC refers to recombinant aPC. aPCincludes and is preferably recombinant human aPC although aPC may alsoinclude other species having protein C proteolytic, amidolytic,esterolytic, and biological (anti-coagulant, anti-inflammatory, orpro-fibrinolytic) activities.

[0033] Human protein C derivative(s) refers to the recombinantlyproduced derivatives of this invention that differ from wild-type humanprotein C but when activated retain the essential properties i.e.,proteolytic, amidolytic, esterolytic, and biological (anti-coagulant,anti-inflammatory, pro-fibrinolytic activities). The definition of humanprotein C derivatives as used herein also includes the activated form ofthe above-identified human protein C derivatives.

[0034] Treating—describes the management and care of a patient for thepurpose of combating a disease, condition, or disorder whether toeliminate the disease, condition, or disorder, or prophylactically toprevent the onset of the symptoms or complications of the disease,condition, or disorder.

[0035] Continuous infusion—continuing substantially uninterrupted theintroduction of a solution or suspension into a vein for a specifiedperiod of time.

[0036] Bolus injection—the injection of a drug in a defined quantity(called a bolus) over a period of time up to about 120 minutes.

[0037] Suitable for administration—a lyophilized formulation or solutionthat is appropriate to be given as a therapeutic agent.

[0038] Unit dosage form—refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

[0039] Hypercoagulable states—excessive coagulability associated withdisseminated intravascular coagulation, pre-thrombotic conditions,activation of coagulation, or congenital or acquired deficiency ofclotting factors such as aPC.

[0040] Protein C deficiency—protein C deficiency as used herein can becongenital or acquired. For either type, the protein C level incirculation is below the lower limit of the normal range. Skilledartisans realize that the normal range is established by a standardprotocol utilizing FDA approved equipment and diagnostic kits fordetermining protein C levels.

[0041] Pharmaceutically effective amount—a therapeutically efficaciousamount of a pharmaceutical compound. The particular dose of the compoundadministered according to this invention will, of course, be determinedby the attending physician evaluating the particular circumstancessurrounding the case, including the compound administered, theparticular condition being treated, the patient characteristics andsimilar considerations.

[0042] Acute coronary syndromes—clinical manifestations of coronaryatherosclerosis complicated by coronary plaque rupture, superimposedcoronary thrombosis, and jeopardized coronary blood flow resulting incoronary ischemia and/or myocardial infarction. The spectrum of acutecoronary syndromes includes unstable angina, non-Q-wave (i.e.,non-ST-segment elevation) myocardial infarction, and Q-wave (i.e.,ST-segment elevation) myocardial infarction.

[0043] Gene Therapy—A therapeutic regime which includes theadministration of a vector containing DNA encoding a therapeuticprotein, directly to affected cells where the therapeutic protein willbe produced. Target tissue for gene delivery include, for example,skeletal muscle, vascular smooth muscle, and liver. Vectors include, forexample, plasmid DNA, liposomes, protein-DNA conjugates, and vectorsbased on adenovirus or herpes virus. Gene therapy has been described,for example, by Kessler et al., PNAS, USA, 93:14082-87, 1996.

[0044] Thrombotic disorders—a disorder relating to, or affected with theformation or presence of a blood clot within a blood vessel. Suchdisorders include, but are not limited to, stroke, abrupt closurefollowing angioplasty or stent placement, and thrombosis as a result ofperipheral vascular surgery.

[0045] Purpura fulminans—ecchymotic skin lesions, fever, hypotensionassociated with bacterial sepsis, viral, bacterial or protozoaninfections. Disseminated intravascular coagulation is usually present.

[0046] Tissue factor pathway inhibitor (TFPI). refers, to, naturally,or, recombinant, forms, of TFPI. This protein is believed to blocktissue-mediated clotting in small blood vessels, which potentially leadsto organ failure and death.

[0047] Serpin—any of a group of structurally related proteins thattypically are serine proteose inhibitors whose inhibiting activity isconferred by a reactive site in C highly variable and mobile peptideloop and that include but are not limited to protein C inhibitor (PCI)and α₁-antitrypsin (α₁-AT).

[0048] Inhibitor recognition sequence S2: the 2^(nd) residue N-terminalto the cleavage site of PCI or α₁-AT.

[0049] Inhibitor recognition sequence S3′: the 3^(rd) residue C-terminalto the cleavage site of PCI or α₁-AT.

[0050] Inhibitor recognition sequence S4′: the 4^(th) residue C-terminalto the cleavage site of PCI or α₁-AT.

[0051] Wild-type protein C—the type of protein C that predominates in anatural population of humans in contrast to that of natural orlaboratory mutant polypeptide forms of protein C.

[0052] Bactericidal permeability increasing protein—includes naturallyand recombinantly produced bactericidal permeability increasing (BPI)protein; natural, synthetic, and recombinant biologically activepolypeptide fragments of BPI protein; biologically active polypeptidevariants of BPI protein or fragments thereof, including hybrid fusionproteins and dimers; biologically active variant analogs of BPI proteinor fragments or variants thereof, including cysteine-substitutedanalogs; and BPI-derived peptides. The complete amino acid sequence ofhuman BPI, as well as the nucleotide sequence of DNA encoding BPI havebeen elucidated by Gray, et al., 1989, J. Biol. Chem 264:9505.Recombinant genes encoding and methods for expression of BPI proteins,including BPI holoprotein and fragments of BPI are disclosed in U.S.Pat. No. 5,198,541, herein incorporated by reference.

[0053] The phrase “in combination with” as used herein, refers to theadministration of additional agents with human aPC derivatives eithersimultaneously, sequentially or a combination thereof. Examples ofadditional agents are anti-platelet agents, thrombolytic agents, and BPIprotein.

[0054] The amino acid abbreviations are accepted by the United StatesPatent and Trademark Office as set forth in 37 C.F.R. 1.822 (d)(1)(1998).

[0055] The present invention provides human protein C derivatives, whichhave increased anti-coagulant activity, resistance to serpininactivation, and increased sensitivity to thrombin activation ascompared to wild-type protein C and the use of these derivatives in thezymogen form as well as in the activated form. The activated form ofhuman protein C derivatives may be produced by activating recombinanthuman protein C derivative zymogen in vitro or by direct secretion ofthe activated form of protein C. The means by which the activationoccurs is not critical and the process aspects of this invention includeany and all means of activation. Human protein C derivatives may beproduced in eukaryotic cells, transgenic animals, or transgenic plants,including, for example, secretion from human kidney 293 cells or AV 12cells as a zymogen, then purified and activated by techniques known tothe skilled artisan.

[0056] Preferred human protein C derivatives of the present inventioninclude S11G:Q32E:N33D:D167F:D172K:L194S,S11G:Q32E:N33D:D167F:D172K:L194S:T254S, S11G:Q32E:N33D:D167F:D172K,andH10Q:S11G:Q32E:N33D:D167F:D172K.

[0057] Human protein C derivative S11G:Q32E:N33D:D167F:D172K:L194Scontains a glycine residue at position 11 instead of the serine residuenormally found at this position, a glutamic acid residue at position 32instead of the glutamine residue normally found at this position anaspartic acid residue at position 33 instead of the asparagine residuenormally found at this position, a phenylalanine at position 167 ratherthan the aspartic acid normally found at this position, a lysine atposition 172 rather than the aspartic acid normally found at thisposition and a serine residue at position 194 instead of the leucineresidue normally found at this position. Other preferred amino acidsubstitutions for positions 194 include Ser, Ala, Thr, His, Leu, Lys,Arg, Asn, Asp, Glu, Gly, and Gln and any amino acid for position 11.

[0058] Human protein C derivative S11G:Q32E:N33D:D167F:D172K:L194S:T254Scontains a glycine residue at position 11 instead of the serine residuenormally found at this position, a glutamic acid residue at position 32instead of the glutamine residue normally found at this position anaspartic acid residue at position 33 instead of the asparagine residuenormally found at this position, a phenylalanine at position 167 ratherthan the aspartic acid normally found at this position, a lysine atposition 172 rather than the aspartic acid normally found at thisposition, a serine residue at position 194 instead of the leucineresidue normally found at this position, and a serine residue atposition 254 instead of the threonine residue normally found at thisposition. Other preferred amino acid substitutions for positions 194 and254 include Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, andGln and any amino acid for position 11.

[0059] Human protein C derivative S11G:Q32E:N33D:D167F:D172K contains aglycine residue at position 11 instead of the serine residue normallyfound at this position, a glutamic acid residue at position 32 insteadof the glutamine residue normally found at this position an asparticacid residue at position 33 instead of the asparagine residue normallyfound at this position, a phenylalanine at position 167 rather than theaspartic acid normally found at this position, and a lysine at position172 rather than the aspartic acid normally found at this position Otherpreferred amino acid substitutions for positions 11 include any aminoacid.

[0060] Human protein C derivative H10Q:S11G:Q32E:N33D:D167F:D172Kpreferably contains a glutamine at position 10 rather than the histidineresidue normally found at this position, a glycine at position 11 ratherthan the serine normally found at this position, a glutamic acid residueat position 32 instead of the glutamine residue normally found at thisposition an aspartic acid residue at position 33 instead of theasparagine residue normally found at this position, a phenylalanine atposition 167 rather than the aspartic acid normally found at thisposition, and a lysine at position 172 rather than the aspartic acidnormally found at this position Other preferred amino acid substitutionsfor positions 11 include any amino acid.

[0061] Other embodiments of the present inventions includeH10Q:S11G:S12K:D167F:D172K:L194S:T254S, S11G:Q32E:D167F:D172K:L194S,S11G:Q32E:D167F:D172K:L194S:T254S, S11G:Q32E:N33F:D167F:D172K:L194S, andS11G:Q32E:N33F:D167F:D172K:L194S:T254S, and activated forms thereofwhich have increased anti-coagulation activity and resistance to serpininactivation, and increased sensitivity to thrombin activation, ascompared to wild-type activated protein C.

[0062] Human protein C derivative H10Q:S11G:S12K:Dl67F:D172K:L194S:T254Spreferably contains a glutamine at position 10 rather than the histidineresidue normally found at this position, a glycine at position 11 ratherthan the serine normally found at this position, a lysine residue atposition 12 rather than a serine residue normally found at thisposition, a phenylalanine at position 167 rather than the aspartic acidnormally found at this position, a lysine at position 172 rather thanthe aspartic acid normally found at this position, a serine at position194 rather than the leucine normally found at this position, and aserine at position 254 instead of a threonine normally found at thisposition. Other preferred amino acid substitutions for positions Otherpreferred amino acid substitutions for positions 194 and 254 includeSer, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, and Gln and anyamino acid for positions 10, 11, and 12.

[0063] Human protein C derivative S11G:Q32E:D167F:D172K:L194S contains aglycine residue at position 11 instead of the serine residue normallyfound at this position, a glutamic acid residue at position 32 insteadof the glutamine residue normally found at this position, aphenylalanine at position 167 rather than the aspartic acid normallyfound at this position, a lysine at position 172 rather than theaspartic acid normally found at this position, and a serine residue atposition 194 instead of the leucine residue normally found at thisposition. Other preferred amino acid substitutions for positions 194include Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, and Glnand any amino acid for position 11.

[0064] Human protein C derivative S11G:Q32E:D167F:D172K:L194S:T254Scontains a glycine residue at position 11 instead of the serine residuenormally found at this position, a glutamic acid residue at position 32instead of the glutamine residue normally found at this position, aphenylalanine at position 167 rather than the aspartic acid normallyfound at this position, a lysine at position 172 rather than theaspartic acid normally found at this position, a serine residue atposition 194 instead of the leucine residue normally found at thisposition, and a serine residue at position 254 instead of the threonineresidue normally found at this position. Other preferred amino acidsubstitutions for positions 194 and 254 include Ser, Ala, Thr, His, Leu,Lys, Arg, Asn, Asp, Glu, Gly, and Gln and any amino acid for position11.

[0065] Human protein C derivative S11G:Q32E:N33F:D167F:D172K:L194Scontains a glycine residue at position 11 instead of the serine residuenormally found at this position, a glutamic acid residue at position 32instead of the glutamine residue normally found at this position aphenyalanine residue at position 33 instead of the asparagine residuenormally found at this position, a phenylalanine at position 167 ratherthan the aspartic acid normally found at this position, a lysine atposition 172 rather than the aspartic acid normally found at thisposition, and a serine residue at position 194 instead of the leucineresidue normally found at this position. Other preferred amino acidsubstitutions for positions 194 include Ser, Ala, Thr, His, Leu, Lys,Arg, Asn, Asp, Glu, Gly, and Gin and any amino acid for position 11.

[0066] Human protein C derivative S11G:Q32E:N33F:D167F:D172K:L194S:T254Scontains a glycine residue at position 11 instead of the serine residuenormally found at this position, a glutamic acid residue at position 32instead of the glutamine residue normally found at this position aphenylalanine residue at position 33 instead of the asparagine residuenormally found at this position, a phenylalanine at position 167 ratherthan the aspartic acid normally found at this position, a lysine atposition 172 rather than the aspartic acid normally found at thisposition, a serine residue at position 194 instead of the leucineresidue normally found at this position, and a serine residue atposition 254 instead of the threonine residue normally found at thisposition. Other preferred amino acid substitutions for positions 194 and254 include Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, andGln and any amino acid for position 11.

[0067] Further embodiments of the present invention include humanprotein C derivatives: S11G:D167F:D172K:L194S,S11G:D167F:D172K:L194S:T254S, S11G:S12K:D167F:D172K:L194S,S12K:D167F:D172K, D167F:D172K:L194S:T254S, S12K:Dl67F:D172K:L194S,S12K:D167F:D172K:L194S:T254S, Q32E:N33D:D167F:D172K,S11G:Q32E:D167F:D172K, S11G:Q32E:N33F:D167F:D172K, and activated formsthereof which have increased anti-coagulant activity, resistance toinactivation by serpins, increased sensitivity to thrombin activation orcombinations of these activities as compared to wild-type humanactivated protein C.

[0068] Human protein C derivative S11G:D167F:D172K:L194S preferablycontains a glycine residue at position 11 rather than a serine residuenormally found at this position, a phenylalanine at position 167 ratherthan the aspartic acid normally found at this position, a lysine atposition 172 rather than the aspartic acid normally found at thisposition, and a serine at position 194 rather than the leucine normallyfound at this position. Other preferred amino acid substitutions forpositions 194 include Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu,Gly, and Gln and any amino acid for position 11.

[0069] Human protein C derivative S11G:D167F:D172K:L194S:T254Spreferably contains a glycine residue at position 11 rather than aserine residue normally found at this position, a phenylalanine atposition 167 rather than the aspartic acid normally found at thisposition, a lysine at position 172 rather than the aspartic acidnormally found at this position, a serine at position 194 rather thanthe leucine normally found at this position, and a serine at position254 instead of a threonine normally found at this position. Otherpreferred amino acid substitutions for positions 194 and 254 includeSer, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, and Gln and anyamino acid for position 11.

[0070] Human protein C derivative S11G:S12K:D167F:D172K:L194S preferablycontains a glycine residue at position 11 rather than a serine residuenormally found at this position, a lysine residue at position 12 ratherthan a serine residue normally found at this position, a phenylalanineat position 167 rather than the aspartic acid normally found at thisposition, a lysine at position 172 rather than the aspartic acidnormally found at this position, and a serine at position 194 ratherthan the leucine normally found at this position. Other preferred aminoacid substitutions for positions 194 include Ser. Ala, Thr, His, Leu,Lys, Arg, Asn, Asp, Glu, Gly, and Gln and any amino acid for position 11and 12.

[0071] Human protein C derivative S12K:D167F:D172K preferably contains alysine residue at position 12 rather than a serine residue normallyfound at this position, a phenylalanine at position 167 rather than theaspartic acid normally found at this position, and a lysine at position172 rather than the aspartic acid normally found at this position. Otherpreferred amino acid substitutions for positions 12 include any aminoacid.

[0072] Human protein C derivative D167F:D172K:L194S:T254S preferablycontains a phenylalanine at position 167 rather than the aspartic acidnormally found at this position, a lysine at position 172 rather thanthe aspartic acid normally found at this position, a serine at position194 rather than the leucine normally found at this position, and aserine at position 254 instead of a threonine normally found at thisposition. Other preferred amino acid substitutions for positions 194,and 254 include Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly,and Gln.

[0073] Human protein C derivative S12K:D167F:D172K:L194S preferablycontains a lysine residue at position 12 rather than a serine residuenormally found at this position, a phenylalanine at position 167 ratherthan the aspartic acid normally found at this position, a lysine atposition 172 rather than the aspartic acid normally found at thisposition, and a serine at position 194 rather than the leucine normallyfound at this position. Other preferred amino acid substitutions forpositions 194 include Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu,Gly, and Gln and any amino acid for position 11.

[0074] Human protein C derivative S12K:D167F:D172K:L194S:T254Spreferably contains a lysine residue at position 12 rather than a serineresidue normally found at this position, a phenylalanine at position 167rather than the aspartic acid normally found at this position, a lysineat position 172 rather than the aspartic acid normally found at thisposition, a serine at position 194 rather than the leucine normallyfound at this position, and a serine at position 254 instead of athreonine normally found at this position. Other preferred amino acidsubstitutions for positions 194 and 254 include Ser, Ala, Thr, His, Leu,Lys, Arg, Asn, Asp, Glu, Gly, and Gln and any amino acid for position12.

[0075] Human protein C derivative Q32E:N33D:D167F:D172K contains aglutamic acid residue at position 32 instead of the glutamine residuenormally found at this position an aspartic acid residue at position 33instead of the asparagine residue normally found at this position, aphenylalanine at position 167 rather than the aspartic acid normallyfound at this position, and a lysine at position 172 rather than theaspartic acid normally found at this position.

[0076] Human protein C derivative S11G:Q32E:D167F:D172K contains aglycine residue at position 11 instead of the serine residue normallyfound at this position, a glutamic acid residue at position 32 insteadof the glutamine residue normally found at this position, aphenylalanine at position 167 rather than the aspartic acid normallyfound at this position, and a lysine at position 172 rather than theaspartic acid normally found at this position. Other preferred aminoacid substitutions for positions 11 include any amino acid

[0077] Human protein C derivative S11G:Q32E:N33F:D167F:D172K contains aglycine residue at position 11 instead of the serine residue normallyfound at this position, a glutamic acid residue at position 32 insteadof the glutamine residue normally found at this position a phenylalanineresidue at position 33 instead of the asparagine residue normally foundat this position, a phenylalanine at position 167 rather than theaspartic acid normally found at this position, and a lysine at position172 rather than the aspartic acid normally found at this position. Otherpreferred amino acid substitutions for position 11 include any aminoacid.

[0078] In addition, human protein C derivatives of the present inventioninclude additional deletions, additions, or substitutions of amino acidresidues of the protein C derivatives described above, but which resultin changes that do not effect the basic characteristics of thisinvention. Amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.Thus, the derivatives of the present invention include derivativeshaving an amino acid sequence that vary from SEQ ID NOS: 3, 4, 5, and 6,by conservative substitutions i.e., those that substitute a residue withanother of like characteristics. Typical substitutions are among Ala,Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp andGlu; among Asn and Gln; and among the basic residues Lys and Arg; oraromatic residues Phe and Tyr. Other derivatives are those in whichseveral, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, oradded in any combination. A preferred embodiment is based on SEQ ID NO:1 includes the addition of the 42 amino acid signal peptide sequence asillustrated in FIG. 1 and shown in SEQ ID NO: 2.

[0079] Preferably, the human protein C derivatives of the presentinvention are not further substituted or modified. That is,substitutions are limited to the derivatives of the present invention.

[0080] The invention also provides DNA compounds for use in making thehuman protein C derivatives. These DNA compounds comprise the codingsequence for the light chain of human protein C zymogen or human proteinC derivative zymogen positioned immediately adjacent to, downstream of,and in translational reading frame with the prepropeptide sequence ofhuman protein C zymogen or human protein C derivative zymogen. The DNAsequences preferably encode the Lys-Arg dipeptide which is processedduring maturation of the protein C molecule, the activation peptide andthe heavy chain of the human protein C derivative. Thus, the humanprotein C derivatives of the present invention are variant or mutantpolypeptides which contain at least 3, preferably 3 to 7 amino acids,which differ from the wild-type protein C sequence identified as SEQ IDNO: 1 (which does not contain the 42 amino acid signal sequence) or thecorresponding wild-type amino acid in SEQ ID NO: 2 (which contains the42 amino acid signal sequence). Thus, one skilled in the art recognizesthat a human protein C derivative which differs from the amino acidsequence of the wild-type protein C sequence identified as SEQ ID NO: 1inherently corresponds to the wild-type protein C sequence identified asSEQ ID NO: 2 at the amino acid position determined after removal of the42 amino acid signal sequence. Furthermore, one skilled in the artrecognizes that prior to activation, the cleavage of the lysine andarginine residues (positions 156 and 157) occurs.

[0081] Those skilled in the art will recognize that, due to thedegeneracy of the genetic code, a variety of DNA compounds can encodethe derivatives described above. U.S. Pat. No. 4,775,624, the entireteaching of which is herein incorporated by reference, discloses thewild-type form of the human protein C molecule. The skilled artisancould readily determine which changes in the DNA sequences which couldencode the exact derivatives as disclosed herein. The invention is notlimited to the specific DNA sequences disclosed. Consequently, theconstruction described below and in the accompanying Examples for thepreferred DNA compounds are merely illustrative and do not limit thescope of the invention.

[0082] All of the DNA compounds of the present invention were preparedby the use of site-directed mutagenesis to change particular positionswithin the human protein C zymogen. The technique for modifyingnucleotide sequences by site-directed mutagenesis is well known to thoseskilled in the art. See e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, second Edition (1989).

[0083] The human protein C derivatives can be made by techniques wellknown in the art utilizing eukaryotic cell lines, transgenic animals, ortransgenic plants. Skilled artisans will readily understand thatappropriate host eukaryotic cell lines include but are not limited toHepG2, LLC-MK₂, CHO-K1, 293, or AV12 cells, examples of which aredescribed in U.S. Pat. No. 5,681,932, herein incorporated by reference.Furthermore, examples of transgenic production of recombinant proteinsare described in U.S. Pat. Nos. 5,589,604 and 5,650,503, hereinincorporated by reference.

[0084] Skilled artisans recognize that a variety of vectors are usefulin the expression of a DNA sequence of interest in a eukaryotic hostcell. Vectors that are suitable for expression in mammalian cellsinclude, but are not limited to: pGT-h, pGT-d; pCDNA 3.0, pCDNA 3.1,pCDNA 3.l+Zeo, and pCDNA 3.1+Hygro (Invitrogen); and, pIRES/Hygro, andpIRES/neo (Clonetech). The preferred vector of the present invention ispIG3 as described in Example 1.

[0085] Other sequences may also be desirable which allow for regulationof expression of the protein sequences relative to the growth of thehost cell. Such regulatory sequences are known to those of skill in theart, and examples include those which cause the expression of a gene tobe turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements may also be present in the vector, for example,enhancer sequences.

[0086] The control sequences and other regulatory sequences may beligated to the coding sequence prior to insertion into a vector, such asthe cloning vectors described above. Alternatively, the coding sequencecan be cloned directly into an expression vector which already containsthe control sequences and an appropriate restriction site.

[0087] In some cases it may be necessary to modify the coding sequenceso that it may be attached to the control sequences with the appropriateorientation; i.e. to maintain the proper reading frame.

[0088] The human protein C derivatives made by any of these methods mustundergo post-translational modifications such as the addition of nine orten gamma-carboxy-glutamates, the addition of oneerythro-beta-hydroxy-Asp (beta-hydroxylation), the addition of fourAsn-linked oligosaccharides (glycosylation) and, the removal of theleader sequence (42 amino acid residues). Such post-translationalmodifications are necessary for efficient production and secretion ofthe protein C derivatives from mammalian cells.

[0089] It is known in the art that post-translational modifications ofrecombinant proteins such as the human protein C derivatives of thepresent invention may vary depending on which host cell line is utilizedfor the expression of the recombinant protein. For example, thepost-translational modification of gamma-carboxylation, which isessential for the anti-coagulant activity of the human protein Cderivatives of the present invention, may be higher, slightly lower, ormuch lower than plasma derived wild-type protein C gamma-carboxylation,depending on the host cell line used (Yan et al., Bio/Technology8(7):655-661, 1990). Such differences in gamma-carboxylation provide abasis for the use of site-directed mutagenesis to change particularpositions within the human protein C molecule that will result in anincrease in anti-coagulant activity.

[0090] The human protein C derivatives of the present invention may beadministered as a zymogen or in the activated form. Methods for theactivation of zymogen forms of human protein C and human protein Cderivatives to activated human protein C and activated human protein Cderivatives are old and well known in the art. Human protein C may beactivated by thrombin alone, by a thrombin/thrombomodulin complex, byRVV-X, a protease from Russell's Viper venom, by pancreatic trypsin orby other proteolytic enzymes.

[0091] The present invention further provides for the treatment of acutecoronary syndromes comprising myocardial infarction, and unstable anginawith human protein C derivatives with increased anti-coagulationactivity, resistance to serpin inactivation, and increased sensitivityto thrombin activation as compared to wild-type aPC.

[0092] The recombinant human protein C derivatives of the presentinvention are also useful for the treatment of thrombotic disorders suchas stroke, abrupt closure following angioplasty or stent placement, andthrombosis as a result of peripheral vascular surgery.

[0093] Additionally, the recombinant human protein C derivatives of thepresent invention are useful for the treatment of vascular occlusivedisorders or hypercoagulable states associated with sepsis, disseminatedintravascular coagulation, major trauma, major surgery, burns, adultrespiratory distress syndrome, transplantations, deep vein thrombosis,heparin-induced thrombocytopenia, sickle cell disease, thalassemia,viral hemorrhagic fever, thrombotic thrombocytopenic purpura, andhemolytic uremic syndrome. In another embodiment, the recombinant humanprotein C derivatives of the present invention are useful for thetreatment of sepsis in combination with bacterial permeabilityincreasing protein. In yet another aspect of this invention theactivated human protein C derivatives of the present invention arecombined with an anti-platelet agent(s) to treat or prevent variousdisorders, such as, thrombotic disease.

[0094] In another embodiment, the recombinant human protein Cderivatives of the present invention are useful for the treatment ofsepsis in combination with tissue factor pathway inhibitor.

[0095] Another aspect of the invention comprises treating the diseasesand conditions caused or resulting from protein C deficiency as definedherein. This aspect of the invention contemplates any and allmodifications to any aPC molecule resulting in increased anti-coagulantactivity and resistance to serpin inactivation as compared to wild-typeaPC.

[0096] The recombinant human protein C derivatives of the presentinvention are useful for the treatment of acute arterial thromboticocclusion, thromboembolism, or stenosis in coronary, cerebral orperipheral arteries or in vascular grafts, in combination with athrombolytic agent such as tissue plasminogen activator, streptokinase,and related compounds or analogs thereof.

[0097] The human protein C derivatives can be formulated according toknown methods to prepare a pharmaceutical composition comprising as theactive agent an aPC derivative and a pharmaceutically acceptable bulkingagent. For example, a desired formulation would be one that is a stablelyophilized product of high purity comprising a bulking agent such assucrose, trehalose or raffinose; a salt such as sodium chloride orpotassium chloride; a buffer such as sodium citrate, Tris acetate, orsodium phosphate, at a pH of about 5.5 to about 6.5; and an activatedhuman protein C derivative.

[0098] The human aPC derivatives of the present invention can beadministered at an appropriate dose level understood and appreciated inthe art and determined by the attending physician evaluating theparticular circumstances surrounding the case. Preferably, the amount ofhuman aPC derivative administered will be from about 0.01 μg/kg/hr toabout 50 4μg/kg/hr. More preferably, the amount of human aPC derivativeadministered will be about 0.1 μg/kg/hr to about 25 μg/kg/hr. Yet evenmore preferably the amount of human aPC derivative administered will beabout 0.1 μg/kg/hr to about 15 μg/kg/hr. Even more preferably the amountof human aPC derivative administered will be about 1 μg/kg/hr to about15 μg/kg/hr. The most preferable amounts of human aPC derivativeadministered will be about 5 μg/kg/hr or about 10 μg/kg/hr.

[0099] Preferably, the human aPC derivatives will be administeredparenterally to ensure delivery into the bloodstream in an effectiveform by injecting a dose of 0.01 mg/kg/day to about 1.0 mg/kg/day, oneto six times a day, for one to ten days. More preferably, the human aPCderivatives will be administered B.I.D. (2 times a day) for three days.

[0100] Alternatively, the human aPC derivatives will be administered ata dose of about 0.01 μug/kg/hr to about 50 μg/kg/hr, by continuousinfusion for 1 to 240 hours.

[0101] The preferred plasma ranges obtained from the amount of humanprotein C derivative administered will be 0.02 ng/ml to less than 100ng/ml.

[0102] In another alternative, the human protein C derivatives will beadministered by injecting a portion (⅓ to ½) of the appropriate dose perhour as a bolus injection over a time from about 5 minutes to about 120minutes, followed by continuous infusion of the appropriate dose for upto 240 hours.

[0103] In another alternative, the human protein C derivatives will beadministered by local delivery through an intracoronary catheter as anadjunct to high-risk angioplasty (with and without stenting, and with orwithout combination therapy with anti-platelet agents). The amount ofhuman protein C derivative administered will be from about 0.01mg/kg/day to about 1.0 mg/kg/day by continuous infusion, bolusinjection, or a combination thereof.

[0104] In another alternative, the human protein C derivatives will beadministered subcutaneously at a dose of 0.01 mg/kg/day to about 10.0mg/kg/day, to ensure a slower release into the bloodstream. Formulationfor subcutaneous preparations will be done using known methods toprepare such pharmaceutical compositions.

[0105] The human protein C derivatives described in this invention haveincreased anti-coagulant activity, resistance to serpin inactivation,and increased sensitivity to thrombin activation. Therefore, thesecompounds provide various advantages over conventional therapeuticagents, for example, site-activation, less frequent administrationand/or smaller dosages, increased efficacy, and thus a reduction in theoverall cost of production of the therapy.

[0106] The following Examples are provided merely to further illustratethe present invention. The scope of the invention shall not be construedas merely consisting of the following Examples.

EXAMPLE 1 Protein C Derivative Construction and Production

[0107] Human protein C derivatives were constructed using the polymerasechain reaction (PCR) following standard methods. The source of thewild-type coding sequence was plasmid pLPC (Bio/Technology 5:1189-1192,1987). The universal PCR primers used include: PC001b;5′GCGATGTCTAGAccaccATGTGGCAGCTCACAAGCCTCCTGC-3′, which encodes for anXbaI restriction site (underlined) used-for subcloning, a Kozakconsensus sequence (lowercase) (Kozak, J Cell Biol 108(2):229-41, 1989),and the 5′ end of the coding region for protein C: PC002e;5′-CAGGGATGATCACTAAGGTGCCCAGCTCTTCTGG-3′, which encodes for the 3′ endof the coding region for human protein C, and includes a BclIrestriction site (underlined) for subcloning. All site-directedmutagenesis was accomplished by established PCR methodology, usingcomplementary oligonucleotides containing the desired sequence changes.The first round of PCR was used to amplify two fragments of the proteinC gene; the 5′ fragment was generated using PC001b and the antisensemutagenic primer, and the 3′ fragment was generated using PC002e and thesense mutagenic primer. The resulting amplified products were purifiedby standard procedures. These fragments were combined and then used as atemplate for a second round of PCR using primers PC001b and PC002e. Thefinal PCR product was digested with XbaI and Bc1I and subcloned intosimilarly digested expression vector pIG3. A wild-type construct wassimilarly generated by PCR using the two universal primers and theplasmid pLPC as the template, followed by subcloning into pIG3. Themutations were confirmed by DNA sequencing of both the coding andnon-coding strands. The pIG3 vector was generated by the insertion of an“internal ribosome entry site” (IRES) (Jackson, et al., Trends BiochemSci 15(12):447-83, 1990) and green fluorescent protein (GFP) (Cormack,et al., Gene 173:33-38, 1996) gene into the mammalian expression vectorpGTD (Gerlitz, et al., Biochem J 295(Pt 1):131-40, 1993). When a cDNA ofinterest is cloned into the multiple cloning site of pIG3, the GBMTpromoter (Berg, et al., Nucleic Acids Res 20(20):5485-6, 1992) drivesexpression of a bicistronic mRNA (5′-cDNA-IRES-GFP-3′). Efficienttranslation of the first cistron is initiated by classical assembly ofribosome subunits on the 5′-methylated cap structure of the mRNA; whilethe normally inefficient translation of a second cistron is overcome bythe IRES sequence which allows for internal ribosome assembly on themRNA. The coupling of the cDNA and reporter on a single mRNA, translatedas separate proteins, allows one to screen for the highest-producingclones on the basis of fluorescence intensity. The expression vectoralso contains an ampicillin resistance cassette for maintenance of theplasmid in E. coli, and a murine DHFR gene with appropriate expressionsequences for selection and amplification purposes in mammalian tissueexpression.

[0108] The adenovirus-transformed Syrian hamster AV12-664 cell line wasgrown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum, 50 μg/mL gentamicin, 200 μg/mL Geneticin (G418), and 10μg/mL vitamin K1. One day prior to transfection, cells were plated at adensity of about 10⁵ cells/25 cm². FspI-linearized plasmids weretransfected using either the calcium phosphate method (ProFection, GibcoBRL-Life Technologies) or FuGene-6 (Boehringer Mannheim), following themanufacturer's instructions. Approximately 48 hours after transfection,the medium was replaced with medium containing 250 nM methotrexate forselection. Colonies resistant to methotrexate were pooled 2-3 weeksafter applying drug selection and expanded. The pools were subjected tofluorescence activated cell sorting based upon GFP fluorescenceintensity (Cormack, 1996), with the most intense 5% of fluorescent cellsbeing retained and expanded. To obtain material for purification,recombinant cells were grown in a modified mixture of Dulbecco'smodified Eagle's and Ham's F-12 media (1:3) containing 1 μg/mL humaninsulin, 1 μg/mL human transferrin, and 10 μg/mL vitamin K1. Conditionedmedia were collected, adjusted to a final concentration of 5 mMbenzamidine and 5 mM EDTA, pH 8.0, and protein C was purified viaanion-exchange chromatography as described (Yan, et al., Bio/Technology8:655-661, 1990). Purified protein was desalted/concentrated inUltrafree-CL 30,000 NMWL filtration units (Millipore) using Buffer A(150 mM NaCl, 20 mM Tris-HCl, pH 7.4), and quantitated by Pierce BCAassay using bovine serum albumin (BSA) as the standard.

EXAMPLE 2 Serpin Resistant Mutants

[0109] The use of site-directed mutagenesis to change particularpositions within human protein C molecule that decrease inactivation byserpins, and consequently result in extended plasma half-lives isdescribed. The recognition sequences in the two primary aPC inhibitorsα₁-AT and PCI reveal some differences that can be exploited by alteringthe residues in aPC that interact with these sequences. Table 1 depictsthe sequences recognized by aPC. The cleavage site occurs between thetwo residues shown in italics. Residues occupying the specific subsites,S2, S3′, and S4′, are underlined.

[0110] In general, the recognized sites in factor Va are different fromthe sites in either factor VIIIa or the inhibitors, therefore, it ispossible to engineer the active site of aPC to preferentially cleave themore critical coagulant factor Va, while at the same time decrease aPC'slikelihood of being inhibited by serpins. TABLE I Coagulation S2...S3.S4. Factors Factor Va 300-313 N C P K K TR N L K KI T R Factor Va500-513 S R S L D RR G I Q RA A A Factor Va 673-685 S T V M A TR K M HDR L E Factor Villa 330-341 P E E P Q LR M K N NE E A Factor Villa560-571 K E S V D QR G N Q IM S D Serpins PCI G T I F T FR S A R LN S Qα₁−AT F L E A I PM S I P PE V K

[0111] In particular, three sites of recognition within the active siteshow distinctive differences between substrate recognition sequences andinhibitor recognition sequences: S2 (the 2^(nd) residue N-terminal tothe cleavage site), S3′ site, and S4′. The S2 site is primarily occupiedby polar residues in the factor Va sequences; unlike PCI and α₁-AT,which have hydrophobic residues at this position. The S3′ site occupiedby polar side chains in all of the substrate sequences, but notably, ahydrophobic side chain in the α₁-AT sequence. The S4′ site is occupiedby charged residues in all three factor Va sequences, but is occupied byhydrophobic residues in the factor VIIIa and inhibitor sequences.

[0112] Based upon the crystal structures of the PPACK-inibited aPC(Mather, et al., EMBO J. 15(24):6822-6831, 1996) and Hirulog 3-inhibitedthrombin (Qiu, et al., Biochemistry 31(47):11689-97, 1992), twoaPC-substrate model structures were created and energy minimized using aCHARMm protocol:

[0113] (1) The sequence representing the factor Va R506 cleavagesequence.

[0114] (2) The recognition sequence of α₁-AT, with the Met substitutedwith Arg (corresponding to a polypeptide of α₁-AT which exhibitsextremely high affinity for aPC).

[0115] These models allowed for the identification of residues whichform critical contacts in these three specific sites. A summary ofresidues which may form specific contacts within the active site, andreplacements that are expected to provide enhanced specificity and/oractivity are summarized in Table II. In general, mutations of residuesthat form contacts within the specific subsites of the active site aredesigned to reflect changes in the environment to drive the specificityof human protein C derivatives away from the recognition of the twoprimary physiological inhibitors, and potentially enhance human proteinC derivative's proteolytic activity. TABLE II Mutations constructed foralteration of specificity aPC Constructed Substrate Site Residuereplacements Contact S2 Thr254 Ser Aliphatic part of sidechain S3′Tyr302 Glu, Gln End of sidechain S4′ Leu194 Ser, Thr, Ala Aliphatic partof sidechain S4′ Ala195 Gly Aliphatic part of sidechain S4′ Leu228 GlnEnd of sidechain S4′ Phe316 Asn Aliphatic part of sidechain

EXAMPLE 3 Activation of Recombinant Protein C

[0116] Complete activation of the zymogen forms of protein C andderivatives was accomplished by incubation with thrombin-sepharose.Thrombin-sepharose was washed extensively with Buffer A. 200 μL ofpacked thrombin-sepharose was mixed with 250 μg of protein C in 1 mL ofthe same buffer and incubated at 37° C. for 4 hours with gentle shakingon a rotating platform. During the course of the incubation, the degreeof protein C activation was monitored by briefly pelleting thethrombin-sepharose, and assaying a small aliquot of the supernatant foraPC activity using the chromogenic substrate S-2366 (DiaPharma).Following complete activation, the thrombin-sepharose was pelleted, andthe supernatant collected. aPC concentration was verified by Pierce BCAassay, and the aPC was either assayed directly, or frozen in aliquots at−80° C. All derivatives were analyzed by SDS-PAGE with eitherCoomassie-blue staining or Western Blot analysis to confirm completeactivation (Laemmli, Nature 227:680-685, 1970).

EXAMPLE 4 Functional Characterization

[0117] The amidolytic activity of recombinant human protein Cderivatives were determined by hydrolysis of the tri-peptide substratesS-2366 (Glu-Pro-Arg-p-nitroanilide), S-2238(Pip-Pro-Arg-p-nitroanilide), and S-2288 (Ile-Pro-Arg-p-nitroanilide).The anti-coagulant activity is shown as measured clotting time in anaPTT at 500 ng mL⁻¹ aPC. Amidolytic activities were measured using thechromogenic substrate S-2366.

[0118] Assays were performed at 25° C., in Buffer A containing 1 mg mL⁻¹BSA, 3 mM CaCl₂, and 0.5 nM aPC. Reactions (200 μL/well) were performedin a 96-well microtiter plate, and amidolytic activity was measured asthe change in absorbance units/min at 405 nm as monitored in a ThermoMaxkinetic micrometer plate reader. Kinetic constants were derived byfitting velocity data at varying substrate concentrations (16 μM to 2μM) to the Michaelis-Menten equation. Changes in A₄₀₅ were converted tommol product using a path length of 0.53 cm (Molecular Devices TechnicalApplications Bulletin 4-1), and an extinction coefficient for thereleased p-nitroanilide of 9620 M-⁻¹ cm⁻¹ (Pfleiderer, Methods Enzymol19:514-521, 1970). Anti-coagulant activity was assessed by measuring theprolongation of clotting time in the activated partial thromboplastintime clotting assay (Helena Laboratories). Clotting reactions weremonitored in a ThermoMax kinetic microtiter plate reader, measuring thetime to Vmax in the change in turbidity.

1 12 1 419 PRT Homo sapiens 1 Ala Asn Ser Phe Leu Glu Glu Leu Arg HisSer Ser Leu Glu Arg Glu 1 5 10 15 Cys Ile Glu Glu Ile Cys Asp Phe GluGlu Ala Lys Glu Ile Phe Gln 20 25 30 Asn Val Asp Asp Thr Leu Ala Phe TrpSer Lys His Val Asp Gly Asp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu HisPro Cys Ala Ser Leu Cys Cys 50 55 60 Gly His Gly Thr Cys Ile Asp Gly IleGly Ser Phe Ser Cys Asp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly Arg PheCys Gln Arg Glu Val Ser Phe Leu 85 90 95 Asn Cys Ser Leu Asp Asn Gly GlyCys Thr His Tyr Cys Leu Glu Glu 100 105 110 Val Gly Trp Arg Arg Cys SerCys Ala Pro Gly Tyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys HisPro Ala Val Lys Phe Pro Cys Gly Arg Pro 130 135 140 Trp Lys Arg Met GluLys Lys Arg Ser His Leu Lys Arg Asp Thr Glu 145 150 155 160 Asp Gln GluAsp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Met Thr 165 170 175 Arg ArgGly Asp Ser Pro Trp Gln Val Val Leu Leu Asp Ser Lys Lys 180 185 190 LysLeu Ala Cys Gly Ala Val Leu Ile His Pro Ser Trp Val Leu Thr 195 200 205Ala Ala His Cys Met Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly 210 215220 Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile 225230 235 240 Lys Glu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Thr AspAsn 245 250 255 Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu SerGln Thr 260 265 270 Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala GluArg Glu Leu 275 280 285 Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly TrpGly Tyr His Ser 290 295 300 Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg ThrPhe Val Leu Asn Phe 305 310 315 320 Ile Lys Ile Pro Val Val Pro His AsnGlu Cys Ser Glu Val Met Ser 325 330 335 Asn Met Val Ser Glu Asn Met LeuCys Ala Gly Ile Leu Gly Asp Arg 340 345 350 Gln Asp Ala Cys Glu Gly AspSer Gly Gly Pro Met Val Ala Ser Phe 355 360 365 His Gly Thr Trp Phe LeuVal Gly Leu Val Ser Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu His AsnTyr Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu 385 390 395 400 Asp Trp IleHis Gly His Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser 405 410 415 Trp AlaPro 2 461 PRT Homo sapiens 2 Met Trp Gln Leu Thr Ser Leu Leu Leu Phe ValAla Thr Trp Gly Ile 1 5 10 15 Ser Gly Thr Pro Ala Pro Leu Asp Ser ValPhe Ser Ser Ser Glu Arg 20 25 30 Ala His Gln Val Leu Arg Ile Arg Lys ArgAla Asn Ser Phe Leu Glu 35 40 45 Glu Leu Arg His Ser Ser Leu Glu Arg GluCys Ile Glu Glu Ile Cys 50 55 60 Asp Phe Glu Glu Ala Lys Glu Ile Phe GlnAsn Val Asp Asp Thr Leu 65 70 75 80 Ala Phe Trp Ser Lys His Val Asp GlyAsp Gln Cys Leu Val Leu Pro 85 90 95 Leu Glu His Pro Cys Ala Ser Leu CysCys Gly His Gly Thr Cys Ile 100 105 110 Asp Gly Ile Gly Ser Phe Ser CysAsp Cys Arg Ser Gly Trp Glu Gly 115 120 125 Arg Phe Cys Gln Arg Glu ValSer Phe Leu Asn Cys Ser Leu Asp Asn 130 135 140 Gly Gly Cys Thr His TyrCys Leu Glu Glu Val Gly Trp Arg Arg Cys 145 150 155 160 Ser Cys Ala ProGly Tyr Lys Leu Gly Asp Asp Leu Leu Gln Cys His 165 170 175 Pro Ala ValLys Phe Pro Cys Gly Arg Pro Trp Lys Arg Met Glu Lys 180 185 190 Lys ArgSer His Leu Lys Arg Asp Thr Glu Asp Gln Glu Asp Gln Val 195 200 205 AspPro Arg Leu Ile Asp Gly Lys Met Thr Arg Arg Gly Asp Ser Pro 210 215 220Trp Gln Val Val Leu Leu Asp Ser Lys Lys Lys Leu Ala Cys Gly Ala 225 230235 240 Val Leu Ile His Pro Ser Trp Val Leu Thr Ala Ala His Cys Met Asp245 250 255 Glu Ser Lys Lys Leu Leu Val Arg Leu Gly Glu Tyr Asp Leu ArgArg 260 265 270 Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile Lys Glu Val PheVal His 275 280 285 Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn Asp Ile AlaLeu Leu His 290 295 300 Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr Ile ValPro Ile Cys Leu 305 310 315 320 Pro Asp Ser Gly Leu Ala Glu Arg Glu LeuAsn Gln Ala Gly Gln Glu 325 330 335 Thr Leu Val Thr Gly Trp Gly Tyr HisSer Ser Arg Glu Lys Glu Ala 340 345 350 Lys Arg Asn Arg Thr Phe Val LeuAsn Phe Ile Lys Ile Pro Val Val 355 360 365 Pro His Asn Glu Cys Ser GluVal Met Ser Asn Met Val Ser Glu Asn 370 375 380 Met Leu Cys Ala Gly IleLeu Gly Asp Arg Gln Asp Ala Cys Glu Gly 385 390 395 400 Asp Ser Gly GlyPro Met Val Ala Ser Phe His Gly Thr Trp Phe Leu 405 410 415 Val Gly LeuVal Ser Trp Gly Glu Gly Cys Gly Leu Leu His Asn Tyr 420 425 430 Gly ValTyr Thr Lys Val Ser Arg Tyr Leu Asp Trp Ile His Gly His 435 440 445 IleArg Asp Lys Glu Ala Pro Gln Lys Ser Trp Ala Pro 450 455 460 3 419 PRTHomo sapiens 3 Ala Asn Ser Phe Leu Glu Glu Leu Arg His Gly Ser Leu GluArg Glu 1 5 10 15 Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys GluIle Phe Glu 20 25 30 Asp Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His ValAsp Gly Asp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala SerLeu Cys Cys 50 55 60 Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe SerCys Asp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg GluVal Ser Phe Leu 85 90 95 Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His TyrCys Leu Glu Glu 100 105 110 Val Gly Trp Arg Arg Cys Ser Cys Ala Pro GlyTyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys His Pro Ala Val LysPhe Pro Cys Gly Arg Pro 130 135 140 Trp Lys Arg Met Glu Lys Lys Arg SerHis Leu Lys Arg Asp Thr Glu 145 150 155 160 Asp Gln Glu Asp Gln Val PhePro Arg Leu Ile Lys Gly Lys Met Thr 165 170 175 Arg Arg Gly Asp Ser ProTrp Gln Val Val Leu Leu Asp Ser Lys Lys 180 185 190 Lys Ser Ala Cys GlyAla Val Leu Ile His Pro Ser Trp Val Leu Thr 195 200 205 Ala Ala His CysMet Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly 210 215 220 Glu Tyr AspLeu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile 225 230 235 240 LysGlu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn 245 250 255Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr 260 265270 Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu 275280 285 Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser290 295 300 Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu AsnPhe 305 310 315 320 Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser GluVal Met Ser 325 330 335 Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly IleLeu Gly Asp Arg 340 345 350 Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly ProMet Val Ala Ser Phe 355 360 365 His Gly Thr Trp Phe Leu Val Gly Leu ValSer Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu His Asn Tyr Gly Val TyrThr Lys Val Ser Arg Tyr Leu 385 390 395 400 Asp Trp Ile His Gly His IleArg Asp Lys Glu Ala Pro Gln Lys Ser 405 410 415 Trp Ala Pro 4 419 PRTHomo sapiens 4 Ala Asn Ser Phe Leu Glu Glu Leu Arg His Gly Ser Leu GluArg Glu 1 5 10 15 Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys GluIle Phe Glu 20 25 30 Asp Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His ValAsp Gly Asp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala SerLeu Cys Cys 50 55 60 Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe SerCys Asp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg GluVal Ser Phe Leu 85 90 95 Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His TyrCys Leu Glu Glu 100 105 110 Val Gly Trp Arg Arg Cys Ser Cys Ala Pro GlyTyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys His Pro Ala Val LysPhe Pro Cys Gly Arg Pro 130 135 140 Trp Lys Arg Met Glu Lys Lys Arg SerHis Leu Lys Arg Asp Thr Glu 145 150 155 160 Asp Gln Glu Asp Gln Val PhePro Arg Leu Ile Lys Gly Lys Met Thr 165 170 175 Arg Arg Gly Asp Ser ProTrp Gln Val Val Leu Leu Asp Ser Lys Lys 180 185 190 Lys Ser Ala Cys GlyAla Val Leu Ile His Pro Ser Trp Val Leu Thr 195 200 205 Ala Ala His CysMet Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly 210 215 220 Glu Tyr AspLeu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile 225 230 235 240 LysGlu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Ser Asp Asn 245 250 255Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr 260 265270 Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu 275280 285 Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser290 295 300 Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu AsnPhe 305 310 315 320 Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser GluVal Met Ser 325 330 335 Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly IleLeu Gly Asp Arg 340 345 350 Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly ProMet Val Ala Ser Phe 355 360 365 His Gly Thr Trp Phe Leu Val Gly Leu ValSer Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu His Asn Tyr Gly Val TyrThr Lys Val Ser Arg Tyr Leu 385 390 395 400 Asp Trp Ile His Gly His IleArg Asp Lys Glu Ala Pro Gln Lys Ser 405 410 415 Trp Ala Pro 5 419 PRTHomo sapiens 5 Ala Asn Ser Phe Leu Glu Glu Leu Arg His Gly Ser Leu GluArg Glu 1 5 10 15 Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys GluIle Phe Glu 20 25 30 Asp Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His ValAsp Gly Asp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala SerLeu Cys Cys 50 55 60 Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe SerCys Asp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg GluVal Ser Phe Leu 85 90 95 Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His TyrCys Leu Glu Glu 100 105 110 Val Gly Trp Arg Arg Cys Ser Cys Ala Pro GlyTyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys His Pro Ala Val LysPhe Pro Cys Gly Arg Pro 130 135 140 Trp Lys Arg Met Glu Lys Lys Arg SerHis Leu Lys Arg Asp Thr Glu 145 150 155 160 Asp Gln Glu Asp Gln Val PhePro Arg Leu Ile Lys Gly Lys Met Thr 165 170 175 Arg Arg Gly Asp Ser ProTrp Gln Val Val Leu Leu Asp Ser Lys Lys 180 185 190 Lys Leu Ala Cys GlyAla Val Leu Ile His Pro Ser Trp Val Leu Thr 195 200 205 Ala Ala His CysMet Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly 210 215 220 Glu Tyr AspLeu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile 225 230 235 240 LysGlu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn 245 250 255Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr 260 265270 Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu 275280 285 Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser290 295 300 Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu AsnPhe 305 310 315 320 Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser GluVal Met Ser 325 330 335 Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly IleLeu Gly Asp Arg 340 345 350 Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly ProMet Val Ala Ser Phe 355 360 365 His Gly Thr Trp Phe Leu Val Gly Leu ValSer Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu His Asn Tyr Gly Val TyrThr Lys Val Ser Arg Tyr Leu 385 390 395 400 Asp Trp Ile His Gly His IleArg Asp Lys Glu Ala Pro Gln Lys Ser 405 410 415 Trp Ala Pro 6 419 PRTHomo sapiens 6 Ala Asn Ser Phe Leu Glu Glu Leu Arg Gln Gly Ser Leu GluArg Glu 1 5 10 15 Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys GluIle Phe Glu 20 25 30 Asp Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His ValAsp Gly Asp 35 40 45 Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala SerLeu Cys Cys 50 55 60 Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe SerCys Asp Cys 65 70 75 80 Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg GluVal Ser Phe Leu 85 90 95 Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His TyrCys Leu Glu Glu 100 105 110 Val Gly Trp Arg Arg Cys Ser Cys Ala Pro GlyTyr Lys Leu Gly Asp 115 120 125 Asp Leu Leu Gln Cys His Pro Ala Val LysPhe Pro Cys Gly Arg Pro 130 135 140 Trp Lys Arg Met Glu Lys Lys Arg SerHis Leu Lys Arg Asp Thr Glu 145 150 155 160 Asp Gln Glu Asp Gln Val PhePro Arg Leu Ile Lys Gly Lys Met Thr 165 170 175 Arg Arg Gly Asp Ser ProTrp Gln Val Val Leu Leu Asp Ser Lys Lys 180 185 190 Lys Leu Ala Cys GlyAla Val Leu Ile His Pro Ser Trp Val Leu Thr 195 200 205 Ala Ala His CysMet Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly 210 215 220 Glu Tyr AspLeu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile 225 230 235 240 LysGlu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn 245 250 255Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr 260 265270 Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu 275280 285 Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser290 295 300 Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu AsnPhe 305 310 315 320 Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser GluVal Met Ser 325 330 335 Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly IleLeu Gly Asp Arg 340 345 350 Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly ProMet Val Ala Ser Phe 355 360 365 His Gly Thr Trp Phe Leu Val Gly Leu ValSer Trp Gly Glu Gly Cys 370 375 380 Gly Leu Leu His Asn Tyr Gly Val TyrThr Lys Val Ser Arg Tyr Leu 385 390 395 400 Asp Trp Ile His Gly His IleArg Asp Lys Glu Ala Pro Gln Lys Ser 405 410 415 Trp Ala Pro 7 1260 DNAHomo sapiens 7 gccaactcct tcctggagga gctccgtcac agcagcctgg agcgggagtgcatagaggag 60 atctgtgact tcgaggaggc caaggaaatt ttccaaaatg tggatgacacactggccttc 120 tggtccaagc acgtcgacgg tgaccagtgc ttggtcttgc ccttggagcacccgtgcgcc 180 agcctgtgct gcgggcacgg cacgtgcatc gacggcatcg gcagcttcagctgcgactgc 240 cgcagcggct gggagggccg cttctgccag cgcgaggtga gcttcctcaattgctcgctg 300 gacaacggcg gctgcacgca ttactgccta gaggaggtgg gctggcggcgctgtagctgt 360 gcgcctggct acaagctggg ggacgacctc ctgcagtgtc accccgcagtgaagttccct 420 tgtgggaggc cctggaagcg gatggagaag aagcgcagtc acctgaaacgagacacagaa 480 gaccaagaag accaagtaga tccgcggctc attgatggga agatgaccaggcggggagac 540 agcccctggc aggtggtcct gctggactca aagaagaagc tggcctgcggggcagtgctc 600 atccacccct cctgggtgct gacagcggcc cactgcatgg atgagtccaagaagctcctt 660 gtcaggcttg gagagtatga cctgcggcgc tgggagaagt gggagctggacctggacatc 720 aaggaggtct tcgtccaccc caactacagc aagagcacca ccgacaatgacatcgcactg 780 ctgcacctgg cccagcccgc caccctctcg cagaccatag tgcccatctgcctcccggac 840 agcggccttg cagagcgcga gctcaatcag gccggccagg agaccctcgtgacgggctgg 900 ggctaccaca gcagccgaga gaaggaggcc aagagaaacc gcaccttcgtcctcaacttc 960 atcaagattc ccgtggtccc gcacaatgag tgcagcgagg tcatgagcaacatggtgtct 1020 gagaacatgc tgtgtgcggg catcctcggg gaccggcagg atgcctgcgagggcgacagt 1080 ggggggccca tggtcgcctc cttccacggc acctggttcc tggtgggcctggtgagctgg 1140 ggtgagggct gtgggctcct tcacaactac ggcgtttaca ccaaagtcagccgctacctc 1200 gactggatcc atgggcacat cagagacaag gaagcccccc agaagagctgggcaccttag 1260 8 1386 DNA Homo sapiens 8 atgtggcagc tcacaagcctcctgctgttc gtggccacct ggggaatttc cggcacacca 60 gctcctcttg actcagtgttctccagcagc gagcgtgccc accaggtgct gcggatccgc 120 aaacgtgcca actccttcctggaggagctc cgtcacagca gcctggagcg ggagtgcata 180 gaggagatct gtgacttcgaggaggccaag gaaattttcc aaaatgtgga tgacacactg 240 gccttctggt ccaagcacgtcgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300 tgcgccagcc tgtgctgcgggcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360 gactgccgca gcggctgggagggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420 tcgctggaca acggcggctgcacgcattac tgcctagagg aggtgggctg gcggcgctgt 480 agctgtgcgc ctggctacaagctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540 ttcccttgtg ggaggccctggaagcggatg gagaagaagc gcagtcacct gaaacgagac 600 acagaagacc aagaagaccaagtagatccg cggctcattg atgggaagat gaccaggcgg 660 ggagacagcc cctggcaggtggtcctgctg gactcaaaga agaagctggc ctgcggggca 720 gtgctcatcc acccctcctgggtgctgaca gcggcccact gcatggatga gtccaagaag 780 ctccttgtca ggcttggagagtatgacctg cggcgctggg agaagtggga gctggacctg 840 gacatcaagg aggtcttcgtccaccccaac tacagcaaga gcaccaccga caatgacatc 900 gcactgctgc acctggcccagcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960 ccggacagcg gccttgcagagcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020 ggctggggct accacagcagccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080 aacttcatca agattcccgtggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140 gtgtctgaga acatgctgtgtgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200 gacagtgggg ggcccatggtcgcctccttc cacggcacct ggttcctggt gggcctggtg 1260 agctggggtg agggctgtgggctccttcac aactacggcg tttacaccaa agtcagccgc 1320 tacctcgact ggatccatgggcacatcaga gacaaggaag ccccccagaa gagctgggca 1380 ccttag 1386 9 1386 DNAHomo sapiens 9 atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttccggcacacca 60 gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgctgcggatccgc 120 aaacgtgcca actccttcct ggaggagctc cgtcacggga gcctggagcgggagtgcata 180 gaggagatct gtgacttcga ggaggccaag gaaattttcg aagatgtggatgacacactg 240 gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgcccttggagcacccg 300 tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcagcttcagctgc 360 gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagcttcctcaattgc 420 tctctggaca acggcggctg cacgcattac tgcctagagg aggtgggctggcggcgctgt 480 agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccccgcagtgaag 540 ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacctgaaacgagac 600 acagaagacc aagaagacca agtattcccg cggctcatta aggggaagatgaccaggcgg 660 ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagtccgcctgcggggca 720 gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatgagtccaagaag 780 ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtgggagctggacctg 840 gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccaccgacaatgacatc 900 gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcccatctgcctc 960 ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagaccctcgtgacg 1020 ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcaccttcgtcctc 1080 aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcatgagcaacatg 1140 gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgcctgcgagggc 1200 gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggtgggcctggtg 1260 agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaaagtcagccgc 1320 tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaagagctgggca 1380 ccttag 1386 10 1386 DNA Homo sapiens 10 atgtggcagctcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60 gctcctcttgactcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120 aaacgtgccaactccttcct ggaggagctc cgtcacggga gcctggagcg ggagtgcata 180 gaggagatctgtgacttcga ggaggccaag gaaattttcg aagatgtgga tgacacactg 240 gccttctggtccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300 tgcgccagcctgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360 gactgccgcagcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420 tctctggacaacggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480 agctgtgcgcctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540 ttcccttgtgggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600 acagaagaccaagaagacca agtattcccg cggctcatta aggggaagat gaccaggcgg 660 ggagacagcccctggcaggt ggtcctgctg gactcaaaga agaagtccgc ctgcggggca 720 gtgctcatccacccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780 ctccttgtcaggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840 gacatcaaggaggtcttcgt ccaccccaac tacagcaaga gcaccagcga caatgacatc 900 gcactgctgcacctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960 ccggacagcggccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020 ggctggggctaccacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080 aacttcatcaagattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140 gtgtctgagaacatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200 gacagtggggggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260 agctggggtgagggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320 tacctcgactggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380 ccttag 138611 1386 DNA Homo sapiens 11 atgtggcagc tcacaagcct cctgctgttc gtggccacctggggaatttc cggcacacca 60 gctcctcttg actcagtgtt ctccagcagc gagcgtgcccaccaggtgct gcggatccgc 120 aaacgtgcca actccttcct ggaggagctc cgtcaagggagcctggagcg ggagtgcata 180 gaggagatct gtgacttcga ggaggccaag gaaattttcgaagatgtgga tgacacactg 240 gccttctggt ccaagcacgt cgacggtgac cagtgcttggtcttgccctt ggagcacccg 300 tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacggcatcggcag cttcagctgc 360 gactgccgca gcggctggga gggccgcttc tgccagcgcgaggtgagctt cctcaattgc 420 tctctggaca acggcggctg cacgcattac tgcctagaggaggtgggctg gcggcgctgt 480 agctgtgcgc ctggctacaa gctgggggac gacctcctgcagtgtcaccc cgcagtgaag 540 ttcccttgtg ggaggccctg gaagcggatg gagaagaagcgcagtcacct gaaacgagac 600 acagaagacc aagaagacca agtattcccg cggctcattaaggggaagat gaccaggcgg 660 ggagacagcc cctggcaggt ggtcctgctg gactcaaagaagaagctggc ctgcggggca 720 gtgctcatcc acccctcctg ggtgctgaca gcggcccactgcatggatga gtccaagaag 780 ctccttgtca ggcttggaga gtatgacctg cggcgctgggagaagtggga gctggacctg 840 gacatcaagg aggtcttcgt ccaccccaac tacagcaagagcaccaccga caatgacatc 900 gcactgctgc acctggccca gcccgccacc ctctcgcagaccatagtgcc catctgcctc 960 ccggacagcg gccttgcaga gcgcgagctc aatcaggccggccaggagac cctcgtgacg 1020 ggctggggct accacagcag ccgagagaag gaggccaagagaaaccgcac cttcgtcctc 1080 aacttcatca agattcccgt ggtcccgcac aatgagtgcagcgaggtcat gagcaacatg 1140 gtgtctgaga acatgctgtg tgcgggcatc ctcggggaccggcaggatgc ctgcgagggc 1200 gacagtgggg ggcccatggt cgcctccttc cacggcacctggttcctggt gggcctggtg 1260 agctggggtg agggctgtgg gctccttcac aactacggcgtttacaccaa agtcagccgc 1320 tacctcgact ggatccatgg gcacatcaga gacaaggaagccccccagaa gagctgggca 1380 ccttag 1386 12 1386 DNA Homo sapiens 12atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120aaacgtgcca actccttcct ggaggagctc cgtcaaggga gcctggagcg ggagtgcata 180gaggagatct gtgacttcga ggaggccaag gaaattttcg aagatgtgga tgacacactg 240gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420tctctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600acagaagacc aagaagacca agtattcccg cggctcatta aggggaagat gaccaggcgg 660ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagctggc ctgcggggca 720gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccaccga caatgacatc 900gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380ccttag 1386

We claim:
 1. A human protein C derivative comprising SEQ ID NO: 1 wherein Asp at position 167 is substituted with Phe; Asp at position 172 is substituted with Lys and further comprising at least one amino acid substitution selected from the group consisting of: His at position 10, Ser at position 11, or Ser at position 12 are independently substituted with any amino acid; Gln at position 32 is substituted with Glu; Asn at position 33 is substituted with Asp or Phe; and, amino acids at positions 194, 195, 228, 249, 254, 302, or 316 are substituted with an amino acid selected from Ser, Ala, Thr, His, Leu, Lys, Arg, Asn, Asp, Glu, Gly, and Gln.
 2. The human protein C derivative of claim 1, wherein said human protein C derivative is in its activated form.
 3. The human protein C derivative of claim 1 wherein Ser at position 11 is substituted with Gly; Gln at position 32 is substituted with Glu; Asn at position 33 is substituted with Asp; and, Leu at position 194 is replaced with Ser (SEQ ID NO: 3).
 4. The human protein C derivative of claim 1 wherein Ser at position 11 is substituted with Gly; Gln at position 32 is substituted with Glu; Asn at position 33 is substituted with Asp; and, Leu at position 194 is replaced with Ser; and Thr at position 254 is replaced with Ser (SEQ ID NO: 4).
 5. The human protein C derivative of claim 1 wherein Ser at position 11 is substituted with Gly; Gln at position 32 is substituted with Glu; and, Asn at position 33 is substituted with Asp (SEQ ID NO: 5).
 6. The human protein C derivative of claim 1 wherein His at position 10 is replaced with Gln; Ser at position 11 is replaced with Gly; Gln at position 32 is substituted with Glu; and, Asn at position 33 is substituted with Asp (SEQ ID NO: 6).
 7. A recombinant DNA molecule encoding the human protein C derivative of any one of claims 1 through
 6. 8. A method of treating acute coronary syndromes and disease states predisposing to thrombosis which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a human protein C derivative of claim
 1. 9. A method of treating vascular occlusive disorders and hypercoagulable states which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a human protein C derivative of claim
 1. 10. A method of treating sepsis which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a human protein C derivative of claim 1 in combination with bactericidal permeability increasing protein or tissue factor pathway inhibitor.
 11. A method of treating thrombotic disorders which comprises: administering to a patient in need thereof a pharmaceutically effective amount of an isolated human protein C derivative of claim 1 in combination with an anti-platelet agent.
 12. A method of treating protein C deficiency which comprises: administering to a patient in need thereof a pharmaceutically effective amount of a human protein C derivative of claim
 1. 13. A method of treating acute arterial thrombotic occlusion, thromboembolism, or stenosis in coronary, cerebral or peripheral arteries or in vascular grafts comprising: administering to a patient in need thereof a pharmaceutically effective amount of a human activated protein C derivative of claim 1 in combination with a thrombolytic agent.
 14. A method of treating human patients with genetically predisposed prothrombotic disorders comprising: administering gene therapy to said patients with a recombinant DNA molecule encoding a protein C derivative of claim
 1. 15. A method of treating thrombotic disorders comprising: administering a human activated protein C derivative of claim 1 by local delivery through an intracoronary catheter.
 16. The method of any one of claims 8 through 17 wherein said human protein C derivative is selected from the group consisting of S11G:Q32E:N33D:D167F:D172K:L194S, S11G:Q32E:N33D!D167F:D172K:L194S:T254S, S11G:Q32E:N33D:D167F:D172K, or H10Q:S11G:Q32E:N33D:D167F:D172K.
 17. A pharmaceutical composition comprising: a human protein C derivative of any one of claims 1 through 6 in a pharmaceutically acceptable diluent.
 18. The pharmaceutical composition of claim 17 wherein said human protein C derivative is selected from the group consisting of S11G:Q32E:N33D:D167F:D172K:L194S, S11G:Q32E:N33D:D167F:D172K:L194S:T254S, S11G:Q32E:N33D:D167F:D172K, or H10Q:S11G:Q32E:N33D:D167F:D172K.
 19. The use of the human activated protein C derivative of claim 1 for the manufacture of a medicament for the treatment of acute coronary syndromes, vascular occlusive disorders and hypercoagulable states, sepsis in combination with bactericidal permeability increasing protein, thrombotic disorders thrombotic dusorders in combination with an anti-platelet agent, genetically predisposed prothrombotic disorders, and sepsis in combination with tissue factor pathway inhibitor.
 20. A vector, comprising a nucleic acid according to claim
 7. 21. A host cell transformed by the vector according to claim
 20. 22. A method of producing a human protein C derivative of claim 1 comprising: (a) transforming a host cell with a vector containing nucleic acid encoding a human protein C derivative; (b) culturing said host cell in a medium appropriate for expression of said human protein C derivative; (c) isolating said human protein C derivative from the culture medium; and, (d) activating said human protein C derivative.
 23. The method according to claim 22 wherein the nucleic acid encodes a human protein C derivative selected from the group consisting of S11G:Q32E:N33D:D167F:D172K:L194S, S11G:Q32E:N33D:D167F:D172K:L194S:T254S, S11G:Q32E:N33D:D167F:D172K, or H10Q:S11G:Q32E:N33D:D167F:D172K.
 24. The method according to claim 22 wherein said host cell is selected from the group consisting of 293 cells and AV12 cells. 